Hematology

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Hematology

stains of blood

and bone marrow

Panoptic methods and methods for diagnostic testing

of leukemia and MDS

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02

03 Panoptic methods

• Sample material • Fixation

• Water quality for wash, rinse and dilution steps - Weise phosphate buffer solution

• Standard staining solutions and dry dye mixtures

- May-Grünwald’s eosin methylene blue - Giemsa’s azure eosin methylene blue - Wright’s eosin methylene blue - Leishman’s eosin methylene blue

• Fast staining method – Hemacolor® for microscopy • Auto-Hemacolor® – Staining set for automatic blood

01 Introduction

Leukemia diagnostics

• Principle of leukemia detection • Sample material

• Fixation – LEUCOGNOST® mixture of fixatives

• Enzyme cytochemical staining

- LEUCOGNOST® ALPA - LEUCOGNOST® EST - LEUCOGNOST® PAS - LEUCOGNOST® POX - LEUCOGNOST® AP - LEUCOGNOST® NASDCL - LEUCOGNOST® Basic kit

Page 24 – 49

Page 6 – 23

Page 5

Content

00 Warning and

Precautions

Page 4

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04

05

06

08 Immersion/Mounting

• Oil immersion

• Mounting with Aquatex®

Storage

• Storage and documentation with Entellan® and Neo-Mount®

Blood cell counting

• Blood cell counting with

- Brilliant Cresyl Blue solution and Brilliant Cresyl Blue ZnCl2 Certistain®

- Türk’s solution

References

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Warning and Precautions

For professional use only

Contraindication

Product must be used in a manner consistent with the instructions set forth in their respective Instructions for Use inserts and brochures. Failure to do so may result in an increased risk of injury to the end user.

Sample preparation

All samples must be treated using state-of-the-art technology. All samples must be clearly labeled. Suitable instruments must be used for taking samples and for their preparation. Follow the manufacturer’s instructions for application/use.

Diagnostics

Suitable controls should be conducted with each application in order to avoid an incorrect result. Further tests must be selected and implemented according to recognized methods. The solution and the dye must be used by the expiry date stated.

Instructions for use

For professional use only

In order to avoid errors, the staining process must be carried out by qualified personnel. National guidelines for work safety and quality assurance must be followed. Microscopes equipped according to the standard must be used.

Protection against infection

Effective measures must be taken to protect against infection.

Instructions for disposal

Used solutions and solutions that are past their shelf-life must be disposed of as special waste in accordance with local guidelines.

Hazard classification

Please observe the hazard classification on the label and the information given in the safety data sheet. The product safety data sheet is available on the Internet and on request.

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Definition of Symbols

Serial number In vitro diagnostic _{medical device} Authorized representative in the _{European Community} Catalogue number Control Caution, consult accompanying _documents Batch code Positive control Contains sufficient for <rt> tests Manufacturer Negative control Temperature limitation

Date of manufacture Sterile Upper limit of temperature Use by (YYYY-MM-DD

or YYYY-MM)

Sterilized using steam or

dry heat Lower limit of temperature Do not reuse Sterilized using ethylene _oxide For IVD performance _{evaluation only} Consult instructions

for use

Sterilized using irradia-tion

Biological risks Sterilized using aseptic _{processing technique}

Introduction

Manufactured by: Merck KGaA Frankfurter Strasse 250 64293 Darmstadt Germany +49 6151 72-0 Distributed by:

EMD Millipore Corporation 290 Concord Rd.

Billerica, MA 01821 USA

1-978-715-4321

Intended Use/Purpose:

These microscopy dyes and chemical solution stains are used for medical purposes, specifically for staining cells and tissues for bacteriology or microbiology

Indication for Use:

For in IVD Diagnostic use

Intended User:

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Panoptic methods

02 | Content

• Sample material

• Fixation

• Water quality for wash, rinse and dilution steps

- Weise phosphate buffer solution

Standard staining solutions and dry dye mixtures

- May-Grünwald’s eosin methylene blue

- Giemsa’s azure eosin methylene blue

- Wright’s eosin methylene blue

- Leishman’s eosin methylene blue

• Fast staining method – Hemacolor® for microscopy

• Auto-Hemacolor® –

Staining set for automatic blood smear staining with the HEMA-TEK* slide stainer

Page

8 8 9 9 10 12 16 18 20 24

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The combination of May-Grünwald’s and Giemsa’s methods for blood smears is described

as panoptic staining and was developed in 1912. Nuclei are stained reddish purple,

plasma of lymphocytes and monocytes bluish, and plasma of the granulocytes light pink.

Panoptic staining can be used for sections and for staining of Spirochetes. Beside

the panoptic method, there are other standard staining methods, which will be presented

here as well.

The typical purple color of cell nuclei, is due to molecular interaction

between eosin Y and an azure B-DNA complex. Both dyes build up the complex.

The intensity of the staining depends on the azure B content and on the ratio azure

B/eosin Y. The staining result can be influenced by several factors such as the pH of

the solutions and buffer solution, buffer substances, fixation, and staining time.

The standard hematological staining solution and dye mixtures all contain eosin Y and

a mixture of methylene blue and the oxidation products. The method characteristical

composition makes the difference in the staining result. The fact is that all available

methods will visualize the sample material in a comparable style. The different methods

are suitable for all types of blood and bone marrow samples. It is more the experience or

the tradition which gives the focus to one specific method.

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Sample material | Fixation

Sample material

Fresh and if possible native sample material should be used for the preparation of blood and bone marrow smears. That is the starting material for all types of staining. The use of anticoagulant as EDTA should be reduced to a minimum. Anticoagulants can reduce the stainability of blood and bone marrow samples and it could be critical especially when the material is used for enzyme cytochemical methods. Blood and bone marrow smears must be dried in the air for at least 30 min and fixed then with the relevant fixative and according to the instruction.

Hematological staining methods are applicable for clinical specimens in cytology as well. Specimen as urine sediment, sputum, fine needle aspiration biopsies (FNAB), imprints, lavages can be processed with these methods in a very good way and belong to the standard application.

Fixation

Methanol is the standard fixative for blood and bone marrow samples. Methanol is a solvent with a long tradition

as fixative in hematology. It reacts fast and is inert e.g. no changes of fine structures will be recognized when the fixation is done with sufficient air dried material. The used methanol should have a concentration of 100 %. A sufficient quality grade should be used to prevent problems with the stainability and the quality of the sample material. Methanol is the gold standard as hematological fixative but it is under discussion because of the strong hazardous classification of the solvent. Methanol has to be used in well ventilated and air-conditioned working places. Safety protection for the staff and the working place must be organized according to the safety instructions. The valid safety data sheet is available in the internet under: www.emdmillipore.com

Ordering information

Product Package size Cat. No.

Methanol

for analysis EMSURE® ACS, ISO, Reag. Ph Eur

1 L 2.5 L

1.06009.1000 1.06009.2500

Method

Fix the air dried slides for 3 min in methanol. The slide can be used directly after the fixation for the relevant staining or the slide can be stored for hours after the fixation. Material which have to be stored longer should placed in a refrigerator.

Physicochemical properties of methanol Chemical and physical data

Ignition temperature 455°C (DIN 51794) Solubility in water (20°C) soluble Melting point -98°C Molar mass 32.04 g/mol Density (20°C) 0.7902 g/cm3

pH (H2O) no data available

Boiling point 64.5°C (1013 hPa) Vapor pressure 128 hPa (20°C) Explosive limits 5.5 – 44 % (V) Flash point 10°C

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Water quality | Weise phosphate buffer solution

Water quality for wash, rinse and dilution steps

Especially hematological staining methods react very sensitive and visible of changes in the quality and pH range of used rinsing solution. Tap water or distilled water is used normally for wash/rinse steps and for dilution of the staining solutions. Tap water has a pH of around 7 and distilled water has a pH < 7, distilled water which is stored for a while react more acid, the pH is lower. Acid water – Eosin Y, an acid dye, reacts more intense when the used water has an acid pH. The staining result is more reddish. Alkaline water – methylene blue and the oxidation products give a reinforcement and more intense azure metachromasy and simultaneously an extenuation of the eosin effect, the bluish-grey shade prevails.

Weise phosphate buffer solution

The pH of the water should be in the range of 6.8 to 7.2. Optimal and reproducible staining results are accomplished by the use of buffered solution. The phosphate buffer tablets according to Weise are especially prepared for the buffering of water for wash, rinse and dilution steps in hematological staining methods. According to the desired staining result, the pH of the used buffer solution is to choose. In the portfolio are offered buffer tablets according to Weise with pH 7.2 and 6.8. We have an extra available – buffer tablet according to Weise with pH 6.4 is used for sample material where a bright orange staining of erythrocytes is required.

Preparation of buffer solution

Dissolve 1 buffer tablet in 1 L distilled water. The tablets are very solid pressed, to keep the content and the buffer strength at the end stable. It take some time to dissolve the tablet in the distilled water. The buffer solution is stable for 4 weeks and should be replaced by a fresh buffer solution after that time.

Buffer tablets acc. to Weise pH 7.2

1 pack (100 tablets)

1.09468.0100

1.11374.0100

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Standard staining solutions and dry dye mixtures

for differential blood and bone marrow smears

Preparation

1. Buffer solution

Dissolve 1 buffer tablet in 1 L distilled water.

2. Dilute May-Grünwald’s solution for manual staining

Dilute 30 mL May-Grünwald’s eosin methylene blue solution with 150 mL distilled water and add 20 mL buffer solution.

3. Dilute May-Grünwald’s solution for staining with staining automat

Slowly add 30 mL buffer solution and 220 mL distilled water to 50 mL May-Grünwald’s eosin methylene blue solution, mix and leave to stand for 10 min.

4. May-Grünwald’s eosin methylene blue solution

Dissolve 0.25 g May-Grünwald’s eosin methylene blue in 100 mL methanol while warming gently on a water bath at 60°C. Stir for 1 h, leave to stand for 24 h and filter.

5. Dilute Giemsa’s solution for manual staining

Dilute 10 mL Giemsa’s azure eosin methylene blue solution with 190 mL buffer solution, mix well, leave to stand for 10 min, and filter if necessary.

Blood smear, May-Grünwald's stain Blood smear, May-Grünwald's stain

May-Grünwald’s eosin methylene blue solution

Procedure

Air-dried smears, fixed in methanol

Staining rack

Reagents Time

May-Grünwald’s solution 3 min Buffer solution (1 mL) add, mix, stain 6 min Rinse with buffer solution 1 min Dry

Staining jar

May-Grünwald’s solution 3 min Dilute May-Grünwald’s solution 6 min Rinse with buffer solution 2 x 1 min Dry

Staining with staining automat

May-Grünwald’s solution 3 min Dilute May-Grünwald’s solution 6 min Buffer solution 1 min Running water (rinse) 2 min Dry 3 min

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Staining – May-Grünwald’s eosin methylene blue

Blood smear, Pappenheim's stain – pH 7.2 Blood smear, Pappenheim's stain – pH 6.8

Results

Cell type May-Grünwald’s Pappenheim’s

Nuclei red to violet purple to violet Lymphocytes plasma blue plasma blue Monocytes plasma dove-blue plasma dove-blue Neutrophilic granulocytes granules light violet granules light violet Eosinophilic granulocytes granules brick-red to red-brown granules brick-red to red-brown Basophilic granulocytes granules dark violet to black granules dark violet to black Thrombocytes violet violet Erythrocytes reddish reddish

May-Grünwald’s eosin methylene blue solution 100 mL 500 mL 1 L 2.5 L 1.01424.0100 1.01424.0500 1.01424.1000 1.01424.2500 May-Grünwald’s eosin methylene blue 25 g 100 g 1.01352.0025 1.01352.0100

Giemsa’s azure eosin methylene blue

100 mL 500 mL

1.09204.0100 1.09204.0500

Pappenheim’s staining: Staining with May-Grünwald’s solution and Giemsa’s solution

Staining of blood and bone marrow smears and clinical-cytological specimens

Staining rack

Cover the smear with 1 mL May-Grünwald's-solution

3 min Add 1 mL buffer solution, mix and stain 3 – 5 min Cover with dilute Giemsa’s solution, stain 15 – 20 min Rinse with buffer solution 1 min Dry

Staining jar

May-Grünwald’s solution 3 – 5 min Dilute Giemsa’s solution 15 – 20 min Rinse with buffer solution 2 x 1 min Dry

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Staining – Giemsa’s azure eosin methylene blue

Blood smear, Giemsa's stain Blood smear, Giemsa's stain

Giemsa’s azure eosin methylene blue solution

Staining of blood and bone marrow smears, paraffin sections and clinical-cytological specimens

Preparation

2. Dilute Giemsa’s solution for manual staining

Dilute 10 mL Giemsa’s azure eosin methylene blue solution with 190 mL buffer solution, mix well, leave to stand for 10 min, and filter if necessary.

3. Dilute Giemsa’s solution for staining with staining automat

Slowly add 25 mL Giemsa’s solution to 275 mL buffer solution, mix and leave to stand for 10 min, and filter if necessary.

4. Giemsa’s azure eosin methylene blue solution

Dissolve 0.76 g Giemsa’s azure eosin methylene blue in 50 mL glycerol and heat for 3 h at 60°C on a water bath, add 50 mL methanol, leave to stand for 5 days and filter.

Procedure

Air-dried smears

Staining rack/Staining jar

Methanol 3 – 5 min Dilute Giemsa’s solution 15 – 20 min Rinse with buffer solution 2 x 1 min Dry

Methanol 3 min Dilute Giemsa’s solution 15 – 20 min Buffer solution 1 min Running water (rinse) 2 min Dry 3 min

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Staining – Giemsa’s azure eosin methylene blue

Blood smear, Giemsa's stain Blood smear, Giemsa's stain

Results with phosphate buffer acc. to Weise pH 6.8 Cell type Giemsa’s staining Pappenheim’s

staining

Nuclei red to violet purple to violet Lymphocytes plasma blue plasma blue Monocytes plasma dove-blue plasma dove-blue Neutrophilic granulocytes granules light violet granules light violet Eosinophilic granulocytes granules red to gray-blue granules brick-red to dark violet Basophilic granulocytes granules dark-violet granules dark violet to black Thrombocytes violet violet Erythrocytes reddish reddish Blood

parasites

nuclei bright red

Pappenheim’s staining – Staining with May-Grünwald’s solution and Giemsa’s solution

Staining rack and staining jar: see May-Grünwald’s solution

May-Grünwald’s solution 4 min Dilute Giemsa’s solution 20 min Rinse with buffer solution 1 min Running water (rinse) 2 min Dry 3 min

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Staining – Giemsa’s azure eosin methylene blue

Giemsa's staining of paraffin sections of bone marrow or other suitable sample material

Step Time

Deparaffinate and rehydrate the sections using standard methods

Distilled water 10 sec. Undiluted, filtered Giemsa's azur eosin

methylene blue solution

15 min 0.1 % acetic acid 10 sec. Distilled water 10 sec. 2-propanol 10 sec. 2-propanol 10 sec. 2-propanol 10 sec. Xylene or Neo-Clear® 5 min Xylene or Neo-Clear® 5 min Use Entellan® new to cover the preparations moistened with xylene and use Neo-Mount® to cover those moistened with Neo-Clear®

Notes on Giemsa's staining of paraffin sections

Always employ separate xylene or Neo-Clear® rinse baths when Giemsa's staining paraffin sections as any ethanol traces in the solutions may result in the preparations being discolored.

Pretreatment of bone marrow and iliac crest biopsy materials:

Optimal results can be achieved using a mild

OSTEOSOFT® decalcifying solution. To gently remove any calcification, the fixed biopsy materials are first placed in OSTEOSOFT® for 6 hours, after which they are transferred to histoprocessing. Smaller blocks are carefully cut and, if required, are again treated with OSTEOSOFT® for an additional 20 minutes.

Results

Cell type Color

Cell nuclei, cells blue, dark blue Collagen, osteoid pale blue Eosinophilic grains red Acidophilic mucopolysaccharides, mastocytes, cartilage matrix

reddish violet Acidophilic materials orange red

Giemsa’s azure eosin methylene blue solution 100 mL 500 mL 1 L 2.5 L 1.09204.0100 1.09204.0500 1.09204.1000 1.09204.2500

Giemsa’s azure eosin methylene blue 25 g 100 g 1.09203.0025 1.09203.0100 May-Grünwald’s eosin methylene blue solution 100 mL 500 mL 1 L 2.5 L 1.01424.0100 1.01424.0500 1.01424.1000 1.01424.2500

Methanol for analysis EMSURE® ACS, ISO, Reag. Ph Eur 1 L 2.5 L 1.06009.1000 1.06009.2500 Glycerol 85 % suitable for use as excipient EMPROVE® exp Ph Eur, BP

1 L 2.5 L

1.04091.1000 1.04091.2500

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Staining – Giemsa’s azure eosin methylene blue

Blood smear, Pappenheim's stain Blood smear, Pappenheim's stain

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Staining – Wright’s eosin methylene blue

Wright’s eosin methylene blue solution

Staining of blood and bone marrow smears and clinical-cytological specimens

Preparation

2. Dilute Wright’s solution for manual staining

Add 20 mL buffer solution and 150 mL distilled water to 30 mL Wright’s eosin methylene blue solution.

3. Dilute Wright’s solution for staining with automated staining

Add 30 mL buffer solution and 220 mL distilled water to 50 mL Wright’s eosin methylene blue solution.

4. Wright’s eosin methylene blue solution

Dissolve 0.25 g Wright’s eosin methylene blue in 100 mL methanol, warm gently on a water bath for 20 – 30 min or until the powder is dissolved, filter before use.

Procedure

Air-dried smears, fixed in methanol

Staining rack

Wright’s solution 1 min Buffer solution (1 mL) add, mix, stain 4 min Rinse with buffer solution 1 min Dry

Staining jar

Wright’s solution 3 min Dilute Wright’s solution 6 min Rinse with buffer solution 2 x 1 min Dry

Wright’s solution 3 min Dilute Wright’s solution 6 min Buffer solution 1 min Running water (rinse) 2 min Dry 3 min

Result

Nuclei red to violet Lymphocytes plasma blue Monocytes plasma gray-blue Neutrophilic granulocytes granules light violet Eosinophilic granulocytes granules brick-red

to red-brown Basophilic granulocytes granules dark violet

to black Thrombocytes violet Erythrocytes reddish

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Staining – Wright’s eosin methylene blue

Blood smear, Wright's stain

Blood smear, Wright's stain Blood smear, Wright's stain

FNAB (Douglas), Wright's stain

Wright’s eosin methylene blue solution 100 mL 500 mL 2.5 L 1.01383.0100 1.01383.0500 1.01383.2500 Wright’s eosin methylene blue 25 g 1.09278.0025

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Staining – Leishman’s eosin methylene blue

Leishman’s eosin methylene blue solution

Staining of blood and bone marrow smears and clinical-cytological specimen

Preparation

2. Dilute Leishman’s solution for manual staining

Dilute 30 mL Leishman’s eosin methylene blue solution with 150 mL distilled water and add 20 mL buffer solution.

3. Dilute Leishman’s solution for staining with an automated stainer

Slowly add 30 mL buffer solution and 220 mL distilled water to 50 mL Leishman’s eosin methylene blue solution, mix and leave to stand for 10 min.

4. Leishman’s eosin methylene blue solution

Dissolve 0.12 g Leishman’s eosin methylene blue in 100 mL methanol while warming gently on a water bath at 40°C, leave 5 days to mature, and filter.

Procedure

Air-dried smears, fixed with methanol

Staining rack

Leishman’s solution 1 min Buffer solution (2 mL) add, mix, stain 5 min Rinse with buffer solution 1 min Dry

Staining jar

Leishman’s solution 3 min Dilute Leishman’s solution 6 min Rinse with buffer solution 2 x 1 min Dry

Leishman’s solution 3 min Dilute Leishman’s solution 6 min Buffer solution 1 min Running water (rinse) 2 min Dry 3 min

Result

Nuclei red to violet Lymphocytes plasma blue Monocytes plasma gray-blue Neutrophilic granulocytes granules light violet Eosinophilic granulocytes granules brick-red to

red-brown Basophilic granulocytes granules dark-violet Thrombocytes violet

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Staining – Leishman’s eosin methylene blue

Blood smear, Leishman's stain Blood smear, Leishman's stain

Leishman’s eosin methylene blue solution 500 mL 1.05387.0500 Leishman’s eosin methylene blue 10 g 1.01350.0010

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Fast staining method – Hemacolor®

Blood smear, Hemacolor® stain Blood smear, Hemacolor® stain

Hemacolor® – Staining set for hematological and clinical specimens

Hemacolor® is a fast staining kit which allows in less then 1 min a staining where the result is brilliant and comparable to a Pappenheim's staining. Hemacolor® contains a fixing solution (methanol), a red (eosin Y) and a blue (methylene blue and azure) staining solution and buffer tablets according to Weise pH 7.2. The fact that the staining solutions are separately applied has the advantage that no dye precipitates occur, which are available in standard hematological staining methods normally. The addition of buffer tablets ensure that the stain is very stable and highly reproducible.

The staining solutions are very stable in use. The buffer solution should be replaced and refreshed after 4 weeks. The slides must be moved in the solution because of the short staining/reaction time it is therefore a requirement to get intense and reproducible results.

Sample material

• Air-dried blood and bone marrow smears

• Clinical specimens in cytology, e.g. urine sediment, sputum, FNAB, imprints, lavages

Preparation

Procedure

Staining in jars

Hemacolor® solution 1 5 x 1 sec. Hemacolor® solution 2 3 x 1 sec. Hemacolor® solution 3 6 x 1 sec. Buffer solution pH 7.2 2 x 10 sec. Dry

Hemacolor® solution 1 30 sec. Hemacolor® solution 2 6 sec. Hemacolor® solution 3 4 sec. Buffer solution pH 7.2 10 sec. Running water (rinse) 10 sec. Dry 3 min

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Fast staining method – Hemacolor®

Body effusion, Hemacolor® stain Tumor imprint, Hemacolor® stain

Staining sets Package size Cat. No.

Hemacolor® staining set 1 pack 1.11674.0001

Kit content:

- Hemacolor® solutions (3 x 100 mL) - Buffer tablets, pH 7.2 (3 tabs)

Hemacolor® staining set 1 pack 1.11661.0001

Kit content:

- Hemacolor® solutions (3 x 500 mL) - Buffer tablets, pH 7.2 (6 tabs)

Single reagents

Hemacolor® solution 1, fixing solution

2.5 L 1.11955.2500

Hemacolor® solution 2, color reagent red

2.5 L 1.11956.2500

Hemacolor® solution 3, color reagent blue

2.5 L 1.11957.2500 Buffer tablets pH 7.2 acc. to Weise 1 pack (100 tablets) 1.09468.0100 Result

Nuclei red to violet Lymphocytes plasma blue Monocytes plasma dove-blue Neutrophilic granulocytes granules light violet Eosinophilic granulocytes granules brick-red

to red-brown Basophilic granulocytes granules dark violet

to black Thrombocytes violet Erythrocytes reddish

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Auto-Hemacolor®

Auto-Hemacolor® – Staining set for automatic blood smear staining

with the HEMA-TEK* slide stainer

Fixation, staining and rinsing of blood smears can be accomplished with these solutions. The methanolic dye solution also fixes the smears. Staining is achieved by the alkaline dye (azure) binding to the acidic builders of the cells, e.g. chromatin, spongioplasm, and the acidic dye (eosin) binding to the alkaline constituents, e.g. cytoplasm.

Sample material

Air-dried blood and bone marrow smears.

Preparation

The staining procedure is prepared by placing the Auto-Hemacolor® staining set (with folding carton) into the automatic staining system.

Insert the Auto-Hemacolor® staining set into the opening provided in the equipment. The pack must fit squarely. Remove the pre-punched cardboard sections of the carton. Insert the suction cannulas into the closures and push them firmly through as far as the protective ring seals on the suction cannulas. Allow the instrument to run in order to remove air bubbles.

* = HEMA-TEK® 2000 slide stainer (Siemens Diagnostics) must be used. Follow the manufacturer’s instructions for installation and service.

Procedure

Place the glass slides with the material downwards to the staining strip.

Air-dried blood smears are transported in a fully automatic flow system (no immersion system) over the staining strip. Pass a carefully measured, fresh quantity of dye, buffer and rinse solutions, in this order, into the capillary space between the slides and the staining strip. The solutions are transferred to the staining strip by adjustable peristaltic pumps.

Technical note

If opened packs are not used for longer than 24 h, the puncture openings of the three reagent vessels must be tightly closed to avoid evaporation, which may result in changes in concentration and formation of precipitates. Remove the cannulas, and clean the cannulas and tubes by placing the cannulas in methanol or ethanol and allowing the equipment to run. Insert the clean and dry cannulas as far as the protective ring seals again, and leave them until the next staining; they usually close tightly.

An automatic sign on the equipment is given when the amount of solution left in the equipment is only sufficient for approx. 20 staining. The sign “stain” is extinguished.

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Auto-Hemacolor®

Blood smear, Auto-Hemacolor® stain Blood smear, Auto-Hemacolor® stain

Auto-Hemacolor® 1 pack 1.15213.0001 Kit content: - Staining solution (200 mL) - Buffer solution, pH 7.0 (480 mL) - Rinse solution, pH 7.0 (950 mL) Result

Auto-Hemacolor® gives a comparable staining result to Giemsa’s staining.

Nuclei violet

Lymphocytes plasma blue-gray Monocytes plasma gray-blue Neutrophilic granulocytes granules red-violet Eosinophilic granulocytes granules red-brown Basophilic granulocytes granules dark-violet Thrombocytes violet

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Leukemia diagnostics

03 | Content

• Principle of leukemia detection

• Sample material

• Fixation – LEUCOGNOST® mixture of fixatives

Enzyme cytochemical staining

- LEUCOGNOST® ALPA - LEUCOGNOST® EST - LEUCOGNOST® PAS - LEUCOGNOST® POX - LEUCOGNOST® AP - LEUCOGNOST® NASDCL

- LEUCOGNOST® Basic kit

• Positive and negative controls

• Classification of immature-cell leukemia

• Myelodysplastic syndrome • HEMATOGNOST Fe®

Page

28 28 29 30 32 34 36 38 40 42 43 44 48 49

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Enzyme cytochemistry – cytochemical staining serves to detect the location and

activity of cellular substances and enzyme systems. In hematology, the PAS,

peroxidase, unspecific and specific esterase and acid phosphatase reactions play a key

role in the classification of leukemia. A strongly reduced alkaline phosphatase index

is characteristic for chronic myeloid leukemia.

These fundamental chromogenic enzyme detection methods were usable in small as

well as in big labs with the use of the LEUCOGNOST® staining kits. The LEUCOGNOST®

staining kits assist in carrying out the semi-quantitative localization and activity

detection of enzyme systems in the cytoplasm of leukemia cells of importance

for differential diagnosis. Thus for instance the tedious weighing out of reagents on a

microbalance is fundamentally avoided. The quantities of substances in all staining

kits are such that all the cytochemical reactions can be carried out without complicated

equipment using commercially available 60 mL jars.

In addition to the LEUCOGNOST® Kits with ready-to-use reagents for the determination

of alkaline leukocyte phosphatase activity as well PAS, peroxidase, specific and

unspecific esterase, and acid phosphatase reactions, all the basic reagents are available

as ready-to-use stock solutions. All the reagents are subject to stringent quality

criteria and undergo a cytochemical function test. Thought the availability of all the

items of the same source and the accompanying charts for the evaluation of the result,

it is possible to achieve a high degree of standardization in the methods.

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Principle of leukemia differentiation | Sample material

Principle of leukemia differentiation

Leukemias are autonomous tumors of the hematopoietic system, mostly of the white blood cell series.

Leukemias are always diagnosed from blood and bone marrow smears panoptically stained according to a hematological standard method. While the recognition of mature leukemia of the chronic lymphatic leukemia type or a chronic myeloid leukemia type is usually unproblematic, cytological fine diagnosis within the group of hemoblastic immature-cell leukemia often causes considerable difficulty. Thus errors in differential diagnosis between acute lymphatic leukemia and acute myeloid leukemia without certain evidence of constant reliable morphological differential criteria such as Auer rods, primary granulation and maturation tendency are unavoidable.

To permit better checking of suspect differential therapeutic effects in the treatment of acute leukemia and to take full advantage of their use if indicated, standardized classification of the hematopoietic immature-cell neoplasias is essential. For over 4 decades, stem-line specific enzyme and substrate detections have been used with the aid of cytochemical stains in the cytoplasms of leukemic blasts. The immunological methods with specific fluorescence labelling of the blastic membranes contribute to a subclassification particularly within the group of acute lymphatic leukemias.

Sample material

Only fresh, native blood and bone marrow smears should be used as the starting material for all stains. The use of EDTA as an anticoagulant for example significantly reduces the peroxidase reaction. In cases where the addition of an anticoagulant is required should the amount reduced on a minimum.

The smears must be dried in the air for at least 30 min and fixed according to the relevant instruction prior to the actual cytochemical reaction.

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Fixative for enzyme cytochemistry

LEUCOGNOST® fixing mixture is especially developed for the fixation of blood and bone marrow smears using the various LEUCOGNOST® kits. LEUCOGNOST® fixing mixture optimally protects enzyme activities, and the reaction times of the different working solutions are specially matched to the fixing mixture.

Only fresh, native blood and/or bone marrow smears should be used as the starting material for all stains. The use of e.g. EDTA as anticoagulant significantly reduces the peroxidase reaction. In any case it is quite unnecessary to add any anticoagulant substances. The thin, air-dried blood and/or bone marrow smears should be stored for maximally 3 days prior to the procedure.

The smears must be dried in air for at least 30 minutes and fixed in LEUCOGNOST® fixing mixture according to the relevant instructions prior to the actual cytochemical reaction.

Procedure

Fix the air-dried blood and bone marrow smears in LEUCOGNOST® fixing mixture 1 – 3 min.

Air-dry and process immediately acc. to the protocol or store at +4 to +8°C until required.

Note: The staining protocols and especially the reaction time in the protocol are associated with the fixation of LEUCOGNOST® fixing mixture.

LEUCOGNOST® fixing mixture

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Staining with cytochemical reagent kit for the diagnosis of leukemia

LEUCOGNOST® ALPA

Detection of the alkaline leukocyte phosphatase activity in leukocytes

The determination of the activity (index) of alkaline leukocyte phosphatase is suitable for the cytochemical differentiation of chronic myeloid leukemia from other diseases of the myeloproliferative type, particularly from myelofibrosis and polycythemia or other inflammatory or tumorous processes. Further, the index of alkaline leukocyte phosphatase represents a simple parameter for prognosis in CML, as it reflects the different phases of activity of the hematological disease.

Principle

Alkaline leucocyte phosphatase (AP) catalyzes the hydrolysis of phosphate esters in alkaline solution. 1-naphthol released from 1-naphthyl phosphate is coupled to a diazonium salt to form a brown azo dye, which is precipitated according to the locality and the AP activity in the cell.

Preparation of staining solution*

Solution A

• Dissolve 4 level measuring spoonful (enclosed = 1.1 g) of reagent 1 in 100 mL distilled water.

Solution B

• Wash the contents of one bottle of reagent 2 into the staining cuvette with 15 mL of solution A.

Solution C

• Wash the contents of one bottle of reagent 3 into a conical flask with 45 mL of solution A, shake vigorously for 2 minutes and filter into the staining cell containing solution B through a full- flow filter.

Mix solutions (A + B + C) well

• The reagent solution is stable for a maximum of 1 1/2 hours. The reagent solution is red brown and rapidly becomes turbid. The turbidity, however, does not influence the staining quality.

Procedure

Steps Time

1. Fix the air-dried blood and bone marrow smears in LEUCOGNOST® fixing mixture

1 – 3 min 2. Wash under running tap water 10 sec. 3. Air dry

4. Place in freshly prepared staining

solution*

10 – 15 min 5. Rinse with distilled water and air dry

6. Stain with Mayer's hemalum solution 5 min 7. Rinse with tap water 1 – 3 min 8. Air dry and cover with Aquatex® and

(29)

Staining – LEUCOGNOST® ALPA

Blood smear, LEUCOGNOST® ALPA Blood smear, LEUCOGNOST® ALPA

LEUCOGNOST® ALPA 1 pack 1.16300.0002

(for 12 staining batches)

Kit content:

- Reagent 1: Tris(hydroxymethyl)-aminomethane - Reagent 2: 1-naphthyl phosphate sodium salt - Reagent 3: Variamin® blue salt B

Result

The brown reaction product is only present in the final mature stages of granulopoiesis. Assess 100 neutrophile ones with segmented nuclei; in the event of neutropenia at the most up to 10 % with rod nuclei. Count according to the degree of staining using the following 5 color intensity steps.

Color intensity steps

0 no reaction

1 single to few granules

2 many granules localized

3 granules diffuse distributed

4 cell completely overcasted with granules

5 maximum number of granules, nucleus frequently no longer visible

Multiply the percentages determined by the factors for the corresponding reaction classes and add the products to obtain the dimension less ALPA index.

Normal range: 10 to 100

A reduced index is pathognomonic for the active disease phase of chronic myeloid leukemia. Only hemolytic anemias, iron deficiency anemias or individual virus diseases produce comparable low index values. Normal and increased values always allow a number of interpretations, so that they are of no significance for differential diagnosis. Chronic myeloid leukemia in remission can also produce normal or even increased ALPA values. In general the index is higher, the more extensively necrobiotic catabolic processes (e.g. inflammatory tissue liquefaction) proceed in inflammatory or tumorous processes.

(30)

Staining – LEUCOGNOST® EST

LEUCOGNOST® EST

Detection of the alpha-naphthyl acetate esterase reaction in leukocytes

Esterase reactions with different substrates facilitate differentiation between myeloblastic and monoblastic leukemia. Apart from the naphthol AS-D chloroacetate esterase reaction, whose reliability is comparable with that of the peroxidase reaction, the 1-naphthyl acetate esterase reaction is the most suitable for identifying monoblastic types of leukemia.

1-Naphthyl acetate esterases accelerate the hydrolytic cleavage of 1-naphthyl acetate to form acetic acid and 1-naphthol, which couples with a diazonium salt to form a red brown azo dye which is insoluble in water.

Solution A

• Dissolve 2 level measuring spoonful (enclosed, 0.8 g) of reagent 1 in 60 mL of distilled water.

Solution B

• Dissolve the contents of 1 bottle of reagent 2 in 2 mL acetone, add to 60 mL of solution A and shake vigorously for 1 minute.

Solution C

• Mix 4 – 5 drops (0.2 mL) of reagent 3 and reagent 4, respectively, in an empty bottle of reagent 2 and wait 1 minute (diazotization time).

Mix solutions B and C and filter through a full flow filter into the staining cell.

Note: The staining solution is stable for a maximum of 2 1/2 hours. The staining must be conducted within 15 minutes after preparing the reagent solution. The staining solutions must be freshly prepared immediately before each staining process.

Procedure

Steps Time

1. Fix the air dried blood and/or bone marrow smears in LEUCOGNOST® fixing mixture

1 – 3 min 2. Wash with distilled water 1 min 3. Place in freshly prepared staining

solution* and incubate in the dark

1 – 2 h 4. Wash with distilled water 10 sec. 5. Stain with Mayer's hemalum solution 30 min 6. Wash under tap water 2 min 7. Air dry and cover with Aquatex® and

a cover glass

The stain is stable for about 5 days without embedding and for only a few hours when covered with immersion oil. The stability can be extended to several months with the use of embedding agent and a cover glass.

(31)

Staining – LEUCOGNOST® EST

Blood smear, LEUCOGNOST® EST Blood smear, LEUCOGNOST® EST

LEUCOGNOST® EST 1 pack 1.16301.0002

Kit content:

- Reagent 1: Phosphate buffer - Reagent 2: 1-naphthyl acetate - Reagent 3: Pararosaniline-HCI solution - Reagent 4: Nitrite solution

Result

1-naphthyl acetate esterase reacts weakly in all hematopoietic cells. In particular monocytes, plasma cells, erythroblasts and megakaryocytes react more strongly. The red-brown granular color reaction in this kit is adjusted such that practically only leukemia monoblasts/monocytes with the highest reactivity become stained.

To classify acute leukemia, determine the percentage of esterase-positive blasts and taking into consideration simultaneous differently graded peroxidase reactions, place in one of the categories below:

Categories

Peroxidase type below 25 % EST-pos. blasts AML, AProL

POX-EST mixed type 25 % – 50 % EST-pos. blasts AMMoL

(32)

Staining – LEUCOGNOST® PAS

LEUCOGNOST® PAS

Detection of the periodic acid Schiff reaction in leukocytes

The PAS reaction is an important method for the identification of lymphatic cell elements. Next to peroxidase and esterase reactions, it is one of the three basic cytochemical staining methods important for differential diagnosis that are regularly carried out in acute cases of leukemia. Smears already stained by the Pappenheim's method can additionally be stained with PAS and the stains subsequently removed with 1 % periodic acid.

Periodic acid cleaves neighboring carbon-carbon bonds in polysaccharides (glycogen) when hydroxyl groups are attached to both carbon atoms. The alcoholic groups are then oxidized to aldehydes, which can subsequently be clearly revealed with Schiff's reagent (fuchsin sulfurous acid), producing a red stain.

Preparation of staining solutions*

Solution A

• Dissolve the contents of 1 bottle of reagent 1 in 60 mL of distilled water and transfer to a staining cuvette.

Solution B

• Dissolve the contents of 1 bottle of reagent 2 in 60 mL of distilled water, transfer to a staining cuvette, add 2 mL of reagent 3 and mix. All the reagent solutions are colorless and stable for 3 hours.

Procedure

Steps Time

1. Fix the air dried blood and bone marrow smears in LEUCOGNOST® fixing mixture

1 – 3 min 2. Wash under running tap water 10 sec. 3. Place in solution A* 30 min 4. Wash with distilled water 10 sec. 5. Place in solution B* 1 min 6. Wash with distilled water 10 sec. 7. Stain in Schiff's reagent (20 to 25°C,

incubate in the dark

30 min 8. Wash in distilled water 10 sec. 9. Place in solution B* 2 min 10. Place in distilled water 3 min 11. Stain with Mayer's hemalum solution 3 min 12. Wash under running tap water 3 – 5 min 13. Air dry and cover with Aquatex® and

a cover glass

The stain is stable for about 30 days without embedding and for only 3 days when covered with immersion oil. The stability can be extended to several months with the use of embedding agent and a cover glass.

(33)

Staining – LEUCOGNOST® PAS

Blood smear, LEUCOGNOST® PAS Blood smear, LEUCOGNOST® PAS Blood smear, LEUCOGNOST® PAS

LEUCOGNOST® PAS 1 pack 1.16302.0002

Kit content:

- Reagent 1: Periodic acid - Reagent 2: Potassium disulfite - Reagent 3: Hydrochloric acid Schiff’s reagent 500 mL

2.5 L

1.09033.0500 1.09033.2500

Result

All polysaccharide and in particular glycogen containing structures are stained bright red. Blast populations that at least partly show a characteristic coarse grained PAS positive granulation generally belong to the lymphatic series. Leukemia blasts of the myeloid series are diffuse to fine grained, sometimes also coarse plaqued and PAS positive. Normal myeloblasts, eosinophilic ones and cells of the unaffected red blood cell series are PAS negative by contrast. Promyelocytes, monocytes, basophilic ones and the entire neutrophilic development series demonstrate a diffuse red coloration that shows up bright red with increasing maturity. Erythroblasts in erythroleukemia and some extremely hyper regenerative anaemias can demonstrate a conspicuous PAS reaction.

(34)

Staining – LEUCOGNOST® POX

LEUCOGNOST® POX

Detection of the peroxidase reaction in leukocytes

The peroxidase reaction, specially the cytochemically significant myeloperoxidase reaction, is used to detect myeloid cell elements, where it is possible to obtain a good estimate of the degree of maturity of the maturing granulocytes from the intensity of the black brown color reaction.

Peroxidase are lysosomal catalases which transfer hydrogen from a suitable donor (previously the carcinogen benzidine, here: 4-chloro-1-naphthol) to a peroxide (here: hydrogen peroxide). The donor 4-chloro-1-naphthol is oxidized and converted to a black brown insoluble dye which can be regarded as an indicator of the peroxidase activity.

Material

Only fresh, native blood and/or bone marrow smears should be used as the starting material for all stains. The use of EDTA as an anticoagulant for example significantly reduces the peroxidase reaction. It is in any case quite unnecessary to add anticoagulant substances. Fine air dried blood and/or bone marrow smears not more than 3 days old are required. The smears must be dried in the air for at least 30 minutes and fixed according to the relevant instructions prior to the actual cytochemical reaction.

Dissolve the contents of 1 bottle of reagent 1 in 15 mL of ethanol and transfer to the staining cuvette. Add with stirring, 45 mL distilled water, 10 drops of reagent 2 and 2 drops of reagent 3.

Note: The reagent solution is colorless and stable for 3 hours.

Procedure

Steps Time

1. Fix the air dried blood or bone marrow smears in LEUCOGNOST® fixing mixture

1 min 2. Wash under running tap water 10 sec. 3. Place in freshly prepared staining

solution*

10 min 4. Rinse with distilled water 10 sec.

Dry in the air

5. Stain with Mayer's hemalum solution 2 min 6. Wash with tap water 3 – 5 min 7. Air dry and cover with Aquatex® and

cover glass

The stain is stable for about 3 days without embedding and for only a few hours when covered with immersion oil. The stability can be extended to several months with the use of embedding agent like Aquatex® and a cover glass.

Result

All cells in the neutrophilic and particularly the eosinophilic series of maturity starting with

promyelocytes have black brown colored granules and are thus clearly peroxidase positive. The more mature myeloblasts also can contain peroxidase positive fermentation islands in their cytoplasms, even in cases in which the Pappenheim's staining method shows primary granulation at the early stage of development. The great majority of normal monocytes also reacts peroxidase positively. Their coloration is, however, significantly weaker than that of the neutrophilic and eosinophilic granulocytes. Basophilic granulocytes and all cells of the lymphatic and erythropoietic series are peroxidase negative.

(35)

Staining – LEUCOGNOST® POX

Blood smear, LEUCOGNOST® POX Blood smear, LEUCOGNOST® POX

LEUCOGNOST® POX 1 pack 1.16303.0002

Kit content:

- Reagent 1: 4-chloro-1-naphthol - Reagent 2:

Tris(hydroxymethyl-aminomethane)-HCI buffer - Reagent 3: Hydrogen peroxide solution Leukemia blast populations which react partly or

completely peroxidase-positively are evidence of acute myeloid leukemia, as the lymphoblasts and lymphoid cells of significance in differential diagnosis are always peroxidase-negative. Auer bodies appear conspicuously strongly. In the absence of a peroxidase reaction, however, acute myeloid leukemia cannot be ruled out from the beginning.

In order to positively identify acute myeloid leukemia with an esterase reaction of less than 50 % positivity as myeloblastic, promyelocytic or myelomonocytic leukemia, the exact percentage of peroxidase positive cells in each blast population must be counted. Additional subdivision of the positive cells according to degree of intensity is not required.

In peroxidase positive leukemia, there are 3 reaction types:

POX type 1

- up to 5 % POX-positive blasts - AML without maturation tendency;

AUL or ALL not excluded

POX type 2

- 5 % to 65 % POX-positive blasts

- AML without maturation tendency or AMMoL

POX type 3

- over 65 % POX-positive blasts

(36)

Staining – LEUCOGNOST® AP

LEUCOGNOST® AP

Detection of the acid phosphatase reaction in leukocytes

Acid phosphatase demonstrates specific activity in almost all hematopoietic cell elements (with the exception of neutrophilic and eosinophilic elements) and this is a particularly pronounced characteristic in T-lymphoblastic cells and plasmocytoma cells.

Acid phosphatase catalyses the hydrolysis of phosphate esters in an acidic medium. Under suitable conditions, naphthol AS-BI is released from naphtho-AS-OL phosphate and coupled with a diazonium salt to give a red brown azo dye which is precipitated in the cell.

Dissolve the following in sequence in 60 mL of distilled water: 2 mL of reagent 1 and 3 level measuring spoonful (enclosed, 0.8 g) of reagent 2.

Mix 4 – 5 drops of reagent 3 and reagent 4, respectively, in a small test tube, wait 1 minute and then add to the solution.

Filter the reagent solution into the staining cuvette through a full flow filter.

Note: The reagent solution is stable for a maximum of 3 1/2 hours. The staining must be conducted within 15 minutes of preparing the reagent solution.

Procedure

Procedure without inhibition by tartrate

Steps Time

1. Fix the air dried blood and bone marrow smears in LEUCOGNOST® fixing mixture

1 – 3 min 2. Wash with distilled water 1 min 3. Place in freshly prepared staining

solution* and incubate in the dark

2 – 3 h 4. Wash with distilled water 10 sec. 5. Stain with Mayer's hemalum solution 15 min 6. Wash under tap water 2 min 7. Air dry and cover with Aquatex® and

a cover glass

The stain is stable for about 10 days without embedding and for only a few hours when covered with immersion oil. The stability can be extended to several months with the use of embedding agent and a cover glass.

Procedure with inhibition by tartrate

The individual reaction steps and solutions are identical with those of the procedure without tartrate inhibition. Only the staining solution is slightly modified by dissolving a further 4 level measuring spoonful (enclosed = 0.35 g) of reagent 5.

(37)

Staining – LEUCOGNOST® AP

Bone marrow biopsy, LEUCOGNOST® AP Blood smear, LEUCOGNOST® AP Blood smear, LEUCOGNOST® AP

LEUCOGNOST® AP 1 pack 1.16304.0002

Kit content:

- Reagent 1: Naphthol AS-OL phosphoric acid - Reagent 2: Sodium acetate

- Reagent 3: Pararosaniline-HCI solution (2 N) - Reagent 4: Nitrite solution 4 %

- Reagent 5: Di-sodium tartrate

Result

In contrast to other lymphatic cell elements, T-lymphoblastic cells demonstrate characteristic red brown fermentation islands. Thus with the aid of acid phosphatase, it is in many cases possible to obtain a clear identification of otherwise cytochemically undifferentiable leukemia.

Addition of tartrate to the reaction mixture inhibits the normal phosphatase activity so that little or no coloration takes place in the blood and bone marrow cells. The acid phosphatase (isoenzyme 5) alone in the characteristic cells of hair cell leukemia is “tartrate resistant” in this procedure and can therefore be used as a diagnostic characteristic.

(38)

LEUCOGNOST® NASDCL

For the detection of naphthol AS-D chloroacetate esterase in leukocytes

Naphthol AS-D chloroacetate esterase brings about the enzymatic hydrolysis of naphthol AS-D chloroacetate to a naphthol compound. This in turn reacts with a diazonium salt to form an insoluble red-violet dye.

Solution A

• Dilute 10 mL of reagent 1 with 60 mL distilled water. Add the contents of bottle 2 and rinse out the bottle 2 – 3 times with a few milliliter of buffer.

Solution B

• Add 15 drops of reagent 3 to bottle 4, mix and allow to incubate for 2 minutes.

Solution C

• Add solution B to solution A and rinse out the bottle 2 – 3 times with a few milliliter of substrate buffer mixture.

Prepare the staining solution immediately prior to use.

Procedure

Fixation

Steps Time

1. Immerse the air-dried smear in LEUCOGNOST® fixing solution

5 min 2. Immerse in distilled water 5 min 3. Air-dry and process immediately or

store at +4 to +8°C until required.

5 min

Staining procedure

Steps Time

1. Incubate in the freshly prepared

staining solution* at room

temperature

30 min 2. Place in distilled water 5 min 3. Counter-stain in Mayer's hemalum 5 min 4. Rinse with tap water 5 min 5. Air-dry and cover with Aquatex® and

cover glass

If the smear has not been mounted, the stain is stable for a few days only; if immersed in oil, the stain is stable for a few hours only. Use of a mounting agent and cover glass prolongs stability to several months.

Result

Naphthol AS-D chloroacetate esterase reacts clearly with both mature and immature granulocytes. Intensive enzyme activity can also be shown in the case of myelocytes, metamyelocytes, stab cells and mast cells. Activity may be detected in myeloblastic leukemia cells, promyelocytes and Auer bodies. Monocytes show such activity only rarely. Eosinophiles, basophiles, megakaryocytes, lymphocytes, plasma cells and red cell precursors show no or at most very weak reaction.

(39)

LEUCOGNOST® NASDCL

Bone marrow biopsy, LEUCOGNOST® NASDCL Bone marrow biopsy, LEUCOGNOST® NASDCL

Blood smear, LEUCOGNOST® NASDCL Blood smear, LEUCOGNOST® NASDCL

LEUCOGNOST® NASDCL 1 pack 1.16198.0001

Kit content:

- Reagent 1: Tris buffer concentrate - Reagent 2: Naphthol AS-D chloroacetate - Reagent 3: Sodium nitrite solution - Reagent 4: Fast Red violet LB salt solution

(40)

Staining – LEUCOGNOST® Basic kit

LEUCOGNOST® Basic kit

The LEUCOGNOST® Basic kit contains reagents that are used with the various LEUCOGNOST® kits and are specially matched in quantity to the kits. These reagents fit perfectly to the single LEUCOGNOST® kits and allow intense and reproducible results. The LEUCOGNOST® Basic kit complete the available LEUCOGNOST® kits.

LEUCOGNOST® Basic kit 1 pack 1.16305.0001

Kit content:

- Reagent 1: Mayer's hemalum (500 mL)

- Reagent 2: LEUCOGNOST® fixing mixture (2 x 500 mL) - Reagent 3: Schiff's reagent (500 mL)

- Reagent 4: Ethanol absolute (500 mL) - Reagent 5: Acetone (20 mL)

(41)

Positive and negative controls

To confirm the result in the face of possible unspecific reactions, it is necessary to conduct a positive and negative control with each cytochemical staining batch.

Positive control

The simplest method here is to simultaneously stain normal blood and bone marrow smears. The cells of these smears with their typical color reactions and color intensities serve as references. In addition, normal cell types in the pathological preparation provide a good internal standard.

Example: A normal blood smear is included in the peroxidase reaction.

Segmented leukocytes must give a strong positive reaction, lymphocytes a negative reaction. If the control gives both these results, it can be assumed that the peroxidase stain was conducted properly.

Negative control

To do this, a second smear from the patient is used. The smear is treated in the same way as the first, except that the actual substrate is left out.

Example: A second smear from the patient is stained alongside the first

in the esterase reaction. In this parallel stain, however, the substrate, alpha-naphthyl acetate, is left out so that no color reaction can take place. If it still takes place, an unspecific reaction is involved which must not be assessed as positive in the first smear either.

(42)

Classification of immature-cell leukemia

Cytochemical stain of normal cells

Cell type POX PAS ALPA AP EST NASDCL

Peroxidase Sudan Black B Periodic acid Schiff reaction Alkaline phosphatase Acid phosphatase Alpha-naphthyl acetate esterase Naphthol AS-D chloroacetate esterase

leukocyte granular ++++ fine granular +++ + to ++++ diffuse (+) (+) +

metamyelocyte granular ++++ fine granular +++ negative to + diffuse (+) negative +

myelocyte granular ++++ fine granular ++ negative to + diffuse + negative ++

promyelocyte granular ++ fine granular + negative to + diffuse + negative +

myeloblast negative to granular +

negative negative negative (+) (+)

eosinophilic eosinophil granules positive

negative to + negative diffuse ++ negative negative

basophilic granular ++++ negative to + negative negative negative negative

monocyte weak granular + negative to + negative + to ++ (+) negative

megakaryocyte negative fine granular + negative diffuse +++ ++++ negative

normoblast negative negative negative ++ ++ negative

lymphocyte negative a few fine to middle +

negative in T cells (+) focal negative

plasma cells negative negative negative diffuse ++ negative

Classification of immature-cell leukemia

Acute leukemia is always diagnosed independently of the total number of cells on the basis of their blast population in panoptically stained blood and bone marrow smears. Further attempts at the differentiation only on basis of cytomorphological criteria are, however, not very reliable. The classification of acute leukemia is therefore heavily based on the stem-line specific enzyme detections within the cytoplasma of leukemic blasts. However, a widely held misunderstanding must be pointed out: cytochemical stains cannot be used as an unselected screening method for better primary detection of acute leukemia.

Cytochemical stain of normal cells

The majority of cases of acute leukemia in adults detected from blasts can be accurately classified from a typical pattern of findings based on PAS, Peroxidase (POX), and alpha-naphthyl acetate esterase (EST) reactions – see table “Cytochemical stain of normal cells” below.

(43)

Classification of immature-cell leukemia

Cytochemical stain of acute leukemia

FAB-Classification POX PAS EST NASDCL AP

Peroxidase Sudan Black Periodic acid Schiff reaction Alpha-naphthyl acetate esterase Naphthol AS-D chloroacetate esterase Acid phosphatase M1 ≥ 3 % + negative to fine granular +

negative negative negative

M2 ++ negative to fine granular + negative + to ++ negative M3, M3 Var +++ negative to fine granular + negative + to ++ negative M4, M4 Eo > 5 % + negative to fine granular + ++ (+) negative M5a, b ± negative to fine granular + +++ ++++ negative

M6 + in myeloic blasts coarse positive (erythropoiesis)

negative negative to (+) negative

M7 negative ± ± negative ±

ALL < 1 % + coarse positive < 5 % negative negative polar positive (T)

AUL negative negative negative negative negative

(positive = focal)

Cytochemical stain of acute leukemia

In general there is no problem in determining to the particular cytochemical subtype and therefore making a differential diagnosis in acute leukemia on basis of the cytochemical constellation in approx. 95 % of cases – see table “Cytochemical stain of acute leukemia” below.

(44)

Classification of immature-cell leukemia

FAB classification of acute leukemia

In approximately 5 % of leukocytes neoplasias with infiltration of bone marrow and release into the blood stream, it is not possible to differentiate between immature lymphatic leukemia with negative PAS reaction and immature myeloid leukemia of the peroxidase -1- type (>/= 5 % POX positive blasts). These cases must be placed into a special group, “cytochemical undifferentiated leukemia”. Sometimes it is possible with the aid of an additional characteristic acid phosphatase reaction and particularly with the aid of immunological determination methods, to reach a fine diagnosis, usually indicating acute lymphatic leukemia.

In this connection attention is drawn to the increasing use of a leukemia classification developed by a French-American-British group of hematologists under the leadership of Bennet (1976), the so-called FAB classification of acute leukemia. It is mainly based on the assessment of comprehensive cytomorphological criteria, the cytochemical findings being allocated only a secondary function.

In accordance with this FAB classification, acute leukemia is divided into 2 main groups with 3 or 6 subgroups. The one main group includes acute lymphoblastic leukemia (ALL) with the subtypes L1 to L3, and the other main group contains acute myeloid leukemia (AML) or acute non-lymphoblastic leukemia (ANLL) with the high inhomogeneous subgroup M1 to M6 including erythroleukemia and megakaryocytic leukemia – see table “FAB classification” on the next page. More recently, the megakaryocytic FAB M7 has been differentiated from FAB M6 erythroleukemia. Nevertheless, even with the FAB classification there is still a not inconsiderable percentage (approx. 3 %) of little differentiated leukemias which cannot easily be placed in the L2 or M1 subgroup. These morphological

and cytochemical non-classifiable cases of acute leukemia are combined according to a new definition in the subgroup M0.

(45)

Classification of immature-cell leukemia

FAB classification of acute leukemia is in correlation to the cytomorphological and cytochemical differentiation characteristics on which they are based.

FAB-Classification Characteristic Cytochemical main reaction Incidence (%) Overall abbreviation

Acute lymphoblastic leukemia (ALL)

L1 small cells (child. L.) PAS, AP 65 ALL

L2 mixed cells, often undiff. PAS + undiff. 30 ALL, AUL

L3 coarse cells, Burkitt type PAS 5 ALL

Acute myeloid leukemia (AML) or acute non-lymphoblastic leukemia (ANLL)

M1 myeloblastic, immature POX-1 14 AML (immature)

M2 myeloblastic, mature POX-2, POX-3, Sudan Black 30 AML (maturing)

M3 promyelocytic, hypergranular POX-3, Sudan Black 6 AProl

M4 myelomonocytic POX-EST, NASDCI 33 AMMoI

M5 monocytic

(A = immature, B = mature)

EST 14 AMoL

M6 erythroblastic, megakaryocytic PAS (erythrobl.) 3 AEL The two most commonly used classification schemata for AML are the older French-American-British (FAB) system

(46)

Myelodysplastic syndrome

Myelodysplastic syndromes (MDS) are diseases that increase with the increase of age population. Thus demographic development indicate significant rise in the number of these disease. Enzyme cytochemical staining techniques can be used in hematological tests for the differentiation of MDS. Beside iron staining, naphthol AS-D chloroacetate esterase (NASDCL) are important methods for MDS diagnosis, as enzyme pattern abnormalities suggestive of MDS can be visualized by both positive and negative reaction.

In refractory anemia, more then 15 % of all nucleated red blood cells are in the bone marrow ringed sideroblasts. Ringed sideroblasts are nucleated red cell precursors which on light microscopy have at least five granules of hemosiderin. The granules are stained blue with the Berlin blue stain/Prussian blue stain for iron. In refractory anemia with excess of blasts (RAEB) may be seen ringed sideroblasts.

MDS diagnosis types: 1. Refractory anemia (RA)

2. Refractory anemia with ringed sideroblasts (RARS, D6)

3. Refractory anemia with excess blasts (RAEB)

4. Chronic myelomonocytic leukemia (CMML)

5. Refractory anemia with excess blasts in transformation (RAEBT)

Sideroblast Ringed sideroblast Double nucleated ringed sideroblast Reticular cell with iron Sideroblast Iron granules in small heaps Ringed sideroblast Coarse iron granules

Refractory anaemia (Ringed sideroblasts)

Tabulae haematologicae,